Type 1 diabetes is an autoimmune disease characterized by T cell- mediated destruction of the insulin-producing cells of the islets of Langerhans. The murine model of insulin dependent diabetes mellitus (IDDM), the non-obese diabetic (NOD) mouse has served as a useful model of the human disease since many of the features of the disease are shared. In both mouse and human the development of diabetes is preceded by heavy islet infiltration with lymphoid cells, including CD4+, CD8+ T cells, dendritic cells and monocytes. Recently it was found that T cell responses to the neuronal enzyme glutamic acid decarboxylase 65 (GAD65) were among the first to be detected in the spleen of young NOD mice, with responses being detected as early as 4 weeks. In addition it was found that if NOD were made tolerant to GAD65, by intra-thymic or intravenous injection, diabetes was prevented. We have recently shown that treatment of NOD mice with dendritic cells pulsed with a single GAD65 peptide can prevent diabetes and that this occurs through the initiation of a GAD65-specific Th2 response. Genetic immunization using either plasmid DNA, depending on the route of immunization, can lead to either a Th1 or Th2 response. We have preliminary evidence in a non-diabetes model that the cellular localization of the transfected protein also profoundly influences the nature of the immune response. Strong Th1 responses, with CTL, are obtained when the protein is cytoplasmic, whereas a Th2 dominated response is seen when the protein is secreted following gene gun immunization of ovalbumin (OVA) cDNA. Genetic immunization can thus be tailored to induce the desired response. In the case of diabetes in the NOD mouse, it has been shown that the induction of Th2 responses to specific GAD65 peptides can lead to a halt in the destructive insulitis and the prevention of diabetes. In this proposal are: 1) To construct GAD65 cDNA plasmids which will allow expression of GAD65 in the cytoplasm, as a secreted protein and as a transmembrane protein; 2) To characterize the immune response initiated following immunization of NOD and non diabetes-prone strains with the three GAD65 constructs; 3) To determine whether DNA immunization GAD65 constructs prevent the initiation of diabetes and insulitis.